In vivo and in vitro studies of transmembrane β-strand deletion, insertion or substitution mutants of the Escherichia coli K-12 maltoporin

被引:14
作者
Charbit, A
Andersen, C
Wang, JA
Schiffler, B
Michel, V
Benz, R
Hofnung, M
机构
[1] Inst Pasteur, Unit Programmat Mol & Toxicol Genet, CNRS, URA 1444, F-75724 Paris 15, France
[2] Univ Wurzburg, Biozentrum, Lehrstuhl Biotechnol, D-97074 Wurzburg, Germany
关键词
D O I
10.1046/j.1365-2958.2000.01748.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
LamB of Escherichia coli K12, also called maltoporin, is an outer membrane protein, which specifically facilitates the diffusion of maltose and maltodextrin through the bacterial outer membrane. Each monomer is composed of an 18-stranded antiparallel beta-barrel. In the present work, on the basis of the known X-ray structure of LamB, the effects of modifications of the beta-barrel domain of maltoporin were studied in vivo and in vitro. We show that: (i) the substitution of the pair of strands beta 13-beta 14 of the E. coli maltoporin with the corresponding pair of strands from the functionally related maltoporin of Salmonella typhimurium yielded a protein active in vivo and in vitro; and (ii) the removal of one pair of beta-strands (deletion beta 13-beta 14) from the E. coli maltoporin, or its replacement by a pair of strands from the general porin OmpF of E. coli, leads to recombinant proteins that lost in vivo maltoporin activities but still kept channel formation and carbohydrate binding in vitro. We also inserted into deletion beta 13-beta 14 the portion of the E. coli LamB protein comprising strands beta 13 to beta 16. This resulted in a protein expected to have 20 beta-strands and which completely lost all LamB-specific activities in vivo and in vitro.
引用
收藏
页码:777 / 790
页数:14
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